Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogens to combat HIV/AIDS. We have taken several important steps over the last two years towards our goal of understanding the structure of trimeric envelope glycoproteins spikes and the structural basis of HIV entry and neutralization. Our work has resulted in the first determination of the structure of trimeric gp120 on the surface of the HIV-1 membrane in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. We demonstrated that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This discovery of the closed and open states of the gp120 trimer has provided a new paradigm to interpret and understand the conformational changes of envelope glycoproteins relevant to viral entry. Extension of these studies to a number of SIV strains is providing new and unexpected insights into the molecular organization of trimeric Env in unliganded states and in complex with both neutralizing and non-neutralizing antibodies. Comparison of over a dozen different HIV and SIV strains shows that most display envelope glycoproteins that are in the closed state of the trimer. However, we have discovered an SIV strain where Env is expressed in a constitutively open state even in the absence of bound CD4. The open conformation suggests that this strain should no longer require CD4 for entry, and indeed this is exactly what we have now been able to demonstrate. CD4-independent viruses are often found in immune-privileged areas of the body such as the central nervous system. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses. In parallel with structural investigation of trimeric Env spikes, we have also initiated systematic efforts to analyze HIV distribution in antigen presenting cells such as macrophages and dendritic cells. HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity and 3D distribution of these compartments has remained controversial. We recently determined the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM we showed the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of 150-200 nm, and lengths of up to 5 mum that extend from vesicular compartments that contain assembling HIV-1 virions to the cell surface. Extension of these studies to dendritic cells has also been informative. The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4+ T-cells and virus-bearing dendritic cells via specialized structures known as virological synapses. Using ion abrasion scanning electron microscopy, electron tomography, and super-resolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T-cells. We demonstrate the striking envelopment of T-cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4+ T-cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T-cells, but virions are not released passively at the synapse;instead virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T-cells from the extracellular milieu, the burial of the site of HIV transfer and the receptor-dependent initiation of virion transfer by T-cells highlight novel aspects of cell-cell HIV transmission, and suggest new approaches for limiting the spread of HIV/AIDS.